Dioxins is a general term for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs). A great number of isomers that differ in the chlorine substitution patterns exist for these three types of skeletal structures. Among the isomers of PCDDs and PCDFs, those having chlorine substituents at the 2-, 3-, 7-, and 8-positions are highly toxic and known to cause dermatitis, multiple neurosis, nystagmus, hepatic insufficiency and like symptoms due to the chlorine substitution.
It is also known that long-term exposure to dioxins even in low concentrations can cause chronic symptoms such as porphyria cutanea tarda and also exhibit various toxic properties such as teratogenicity and carcinogenicity.
Furthermore, in recent years, so-called “endocrine disruptors” having the action of disrupting endocrine functions in humans and wild animals have been the focus of attention as a global environmental problem. It has also been revealed that dioxins might be an endocrine disruptor having estrogen activity.
It has become clear that dioxins with their various toxic properties are contained in chemicals such as herbicides and insecticides, exhaust gases and fly ashes from garbage incineration facilities, waste water from papermills, etc. Dioxins are thus detected not only in environmental samples from the air, soils, waters and sediments of rivers, harbors and ports around big cities, etc. but also in biological samples such as foods, blood, urine and mothers' milk. Since such a widespread contamination of the environment has been a big social problem, there is a pressing need to know the amount of dioxin exposure in the environment.
The measurement of dioxins requires high-precision analytical data. Therefore, official methods that comprise extracting, concentrating and purifying dioxins using various chromatographic techniques and subsequent analysis using an expensive analyzer such as a high resolution gas chromatography/mass spectrometer are conventionally used. Such analytical methods are highly sensitive and capable of multi-component analysis so that two or more compounds can be identified and quantitatively determined at one time. On the other hand, such methods have problems in that capital investment such as expensive special equipment and clean rooms is necessary and specialists who are skilled in analysis are required and it takes a long time to obtain results because of the complicated pretreatment.
For such reasons, the development of a highly sensitive, easy dioxin immunoassay method is desired. Environmental pollutant detection techniques using an antibody immunoassay have attracted attention to achieve this object.
Immunoassay is a method for detecting or quantitatively determining a trace amount of an antigen using the capacity of an antibody to specifically recognize the antigen, whereby the antigen can be measured with high sensitivity due to the high affinity and high specificity of the antibody for the antigen. Thus immunoassay has various merits such as simplicity in pretreatment of samples, easy and quick measurement of multiple samples and low cost per measurement, and has been used in a wide variety of fields such as medicine, biochemistry, pharmaceutical sciences and agriculture. To detect or quantitatively determine the targeted substance by immunoassay, it is necessary to label the antibody or antigen, and various labeling methods have been developed. Due to its simplicity, enzyme immunoassay (EIA) using an enzyme has widely been used in clinical tests and the biochemistry field to quantitatively determine targeted substances in biological samples. EIA can be roughly classified into competitive assays and non-competitive assays, based on the form of antigen-antibody interaction. Low molecular weight compounds such as dioxins are usually determined by competitive assays.
In EIA, the concentration of a targeted compound in a sample is calculated from a standard curve that is prepared by using the same compound as the targeted compound as a standard and quantitatively determining the standard in the same manner as the sample. However, since dioxins encompass isomers of three types of compounds having different skeletal structures, the isomers being different in the chlorine substitution pattern, it is difficult to decide which compound should be used as a standard.
Since the toxicity of dioxins differs among congeners and isomers, the toxicity level of dioxins, a mixture of congeners and isomers having different toxicities, varies depending on the ratio of the constituent isomers. A simple sum of the individual isomer amounts does not accurately represent dioxin toxicity.
Many of the dioxin EIA systems so far developed utilize the most toxic 2,3,7,8-tetrachlorobenzo-p-dioxin (2,3,7,8-TeCDD) as a standard (Anal. Chem. 70, 1092–1099). The toxicity of individual dioxin isomers is expressed by toxic equivalency factors (TEFs), which are relative toxicity factors of the isomers compared to 2,3,7,8-TeCDD which is set as 1. The concentrations of the individual isomers are multiplied by their TEFs to calculate their toxicity. The sum of these toxicity values is the toxic equivalent (TEQ), i.e., the total toxicity of all the isomers existing in the target.
Thus the EIA system, which mainly measures 2,3,7,8-TeCDD, can not be said to be an accurate measurement system for dioxin toxicity. In particular, dioxin TEQ of exhaust gas emitted from waste incinerators, which are a main source of dioxins, is known to be highly correlated to the concentration of pentachlorinated dibenzofurans or hexachlorlnated dibenzofurans rather than 2,3,7,8-TeCDD. In addition, when the analytes are exhaust gas samples, there are cases in which the measurements obtained using the conventional EIA system are significantly different from the results of instrumental analysis. Thus the use of the EIA system may be limited.
Moreover, when 2,3,7,8-TeCDD or like dioxin isomers are used as a standard in EIA, the assayer should handle a highly toxic compound for the standard preparation, resulting in the necessity of ensuring safety and involving problems such as the mental burden on the assayer.
Therefore, a non-toxic compound is desired as a standard for the quantitative determination of dioxins in samples. In this connection, Japanese Unexamined Patent Publications Nos. 128731/2002, 131316/2002 and 155023/2002 describe using a chlorophenol derivative as a standard.